Analyzes of Molecular Mechanism of Protein Secretion Using Abnormal Protein C as a Model Protein

1.五种重组蛋白C Arg 15突变体的分泌分析：蛋白C（PC）缺乏分为两种类型. I型显示酶活性和抗原浓度的同等降低，II型仅显示功能活性的降低。在整个PC分子中已经鉴定出40多个突变，其中大多数是由通过单个碱基交换的单个氨基酸替换引起的。在这些突变中，Gla结构域中Arg 15被Gly取代被称为II型突变（PC-Yonago），而Arg-15到Trp的替换被报道为I型或II型，并且Arg-15到Gln的突变被报道为I型。这些表明第15位的氨基酸突变影响PC的分泌和功能。我们采用PCR盒突变法将Arg-15突变为一个碱基可变的氨基酸，即G、W、Q、L或P，并构建了pcD 2-SRa-PC表达载体。将这些载体瞬时转染C 0 关于我们 S-7细胞（2x10^5个细胞），磷酸钙共沉淀。通过ELISA（各4次实验）测定细胞内和分泌部分的重组PC抗原量。细胞内和分泌组分中野生型rPC的量分别为8.4和131.3 ng/皿/48 h。R15 G突变体的相对含量为133.3？B130.8%（细胞内）和67.1？B15.9%（分泌型）的野生型。这表明R15 G突变体的表达和分泌几乎与正常相等，这与该病例的表现形式II型缺陷一致。R15 W突变体的相对含量为55.9？B16.1%（细胞内）和0.4？B10.1%（分泌型），这强烈表明该突变导致I型缺陷。R15 Q突变体的表型为92.2？B16.2%（细胞内）和75.4？B113.3%（分泌型）。与R15 Q的I型表现不同，重组体是分泌型的。R15 L和R15 P突变体在培养基中的分泌量也分别为野生型的54.1%和55.0%，因此，这两个未被识别的突变可能导致II型缺陷。来自人（293细胞）和仓鼠（BHK-21/C13细胞）的其它肾细胞系也显示出与COS-7细胞类似的趋势。Warferin处理的蛋白C的分泌分析：Warferin是一种维生素K的拮抗剂，已知它能破坏微粒体维生素K循环，导致蛋白C和其它一些维生素K依赖性凝血因子的血浆水平降低。我们研究了华法林对293或BHK细胞表达的重组蛋白C分泌的影响，以及对HepG 2细胞内源性分泌的影响。与维生素K处理过的细胞相比，Warfatin导致蛋白C分泌量减少2 - 4倍。使用三种细胞系的脉冲追踪实验表明，虽然蛋白C在维生素K的存在下完全分泌，但华法林处理的细胞中放射性总量的减少表明细胞内降解。这种降解依赖于华法林的浓度，并且不受内质网（ER）-高尔基体转运抑制剂（布雷菲德菌素A）或lysosonotropic抑制剂的抑制，这表明降解发生在前高尔基体，非溶酶体隔室。在测试的蛋白酶抑制剂中，N-乙酰基-Leu-Leu-蛋氨酸和N-乙酰基-Leu-Leu-正亮氨酸以剂量依赖性方式阻断在warafarin存在下合成的蛋白C前体的降解，并且前体在细胞内积累。然而，这两种抑制剂都没有干扰维生素K处理过的细胞的分泌。因此，半胱氨酸蛋白酶似乎是降解的原因。细胞内葡萄糖苷酶I和II，或甘露糖苷酶的抑制剂没有表现出对降解的影响。在华法林存在下合成的蛋白C与蛋白二硫键异构酶（ER驻留蛋白）位于同一细胞器中，并且细胞内蛋白C对内切糖苷酶H消化敏感。此外，使用DPS的交联实验表明，在华法林处理的细胞中，细胞内蛋白C前体与100 kDa、80 kDa、40 kDa和35 kDa的蛋白相关联。从这些结果中，我们得出结论，在华法林的存在下合成的蛋白C被选择性降解的半胱氨酸蛋白酶（S）在ER，通过“质量控制”的机制。少

1. ANALYSIS OF SECRETION OF FIVE RECOMBINANT ARG 15 MUTANTS OF PROTEIN C :Protein C (PC) deficiency is classified into two types. Type I shows equivalent reductions of both enzymatic activity and antigen concentration and type II shows only reduction of functional activity. More than 40 mutations have been identified in all over the PC molecule and most of them are caused by a single amino acid replacement through a single base exchange. Among these mutations, the replacement of Arg 15 to Gly in the Gla-domain is known as type II (PC-Yonago) , while the Arg-15 to Trp replacement was reported as either type I or II,and the Arg-15 to Gln mutation as type I.These indicate that amino acid mutations at the 15th position affect both the secretion and function of PC.To elucidate this hypothesis, we mutated Arg-15 to one base changeable amino acids, i.e.G,W,Q,L,or P by the PCR cassette mutation method and constructed pcD2-SRa-PC expression vectors. These vectors were transfected transiently CO … More S-7 cells (2x10^5 cells) by calcium phosphate coprecipitation. The recombinant PC antigen amounts of intracellular and secreted fractions were determined by ELISA (4experiments each) . The amounts of wild type rPC in the intracellular and secreted fractions were 8.4 and 131.3 ng/dish/48h, respectively. The relative amounts of R15G mutant were determined as 133.3? B130.8% (intracellular) and 67.1? B15.9% (secreted) of those of wild type. This suggests that R15G mutant was expressed and secreted nearly equal to normal, which agrees with type II deficiency of the manifestation of this case. The relative amounts of R15W mutant were 55.9? B16.1% (intracellular) and 0.4? B10.1% (secreted) , which strongly suggests that this mutation causes type I deficiency. The R15Q mutant showed 92.2? B16.2% (intracellular) and 75.4? B113.3% (secreted) . Unlike type I manifestation of R15Q,the recombinant was secretory. The R15L and R15P mutants were also secreted by 54.1 and 55.0%, respectively, of that of wild type in the medium.Thus, these two unrecognized mutations may be predicted to cause type II deficiency. Other kidney cell lines derived from human (293 cells) and hamster (BHK-21/C13 cells) also showed similar tendency with that of COS-7 cells.ANALYSIS OF SECRETION OF WARFERIN-TREATED PROTEIN C :Warfarin, an antagonist of vitamin K,is known to disrupt a microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C as well as some other vitamin K-dependent coagulation factors. We studied the effect of warfarin on secretion of recombinant protein C expressed in 293 or BHK cells, or on the endogenous secretion from HepG2 cells. Warfarin caused a two-to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using three cell lines showed that, although protein C was fully secreted in the presence of vitamin K,the decrease in the total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER) -Golgi transport inhibitor (brefeldin A) nor lysosonotropic inhibitors, suggesting that the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warefarin and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion from vitamin K-treated cells. Thus, a cysteine protease (s) appeared to be responsible for the degradation. Inhibitors for intracellular glucosidase I and II,or for mannosidase showed no effect on the degradation. Protein C synthesized in the presence of warfarin was located in the same organelle with protein disulfide isomerase, an ER-resident protein, and the intracellular protein C was sensitive to endoglycosidase H digestion. Moreover, cross-link experiments using DPS suggested that intracellular protein C precursor associated with 100kDa, 80kDa, 40kDa, and 35kDa proteins in the warfarin-treated cells. From these results we conclude that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease (s) in the ER,through a "quality control" mechanism. Less